ECM modulated early kidney development in embryonic organ culture

Abstract

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases.

Introduction

Prospects for the treatment of severely injured kidneys, kidney failure or chronic kidney disease are currently limited. Therefore, new therapies to recover renal function are an important clinical objective motivating research for regenerative medical strategies. Most of these approaches rely on the use of stem and progenitor cells, which are either directly injected [1], [2], or transplanted as pre-differentiated tissue [3], [4], [5], [6], [7]. Regenerative therapies require an in-depth understanding of development, maintenance, turnover and repair of the respective organ. Organ and tissue culture techniques are increasingly receiving attention and are used to cultivate material from both embryonic and adult origin [8], [9], [10], [11]. In contrast to conventional cell culture, were only one or few cell types are cultured simultaneously, these approaches can provide additional insights into the development of organs and tissues. As a specific cluster of different cells with their specialized ECM is explanted for the use in organ or tissue culture, the in vivo situation is resembled for the most part while these approaches are still in vitro methods with the advantage of easy experimental accessibility.

Tissue-specific cellular microenvironments influence direct cellular fate and function [12], [13]. Physical and (bio)chemical signals trigger different signaling pathways of cells [14], [15], [16], [17]. These processes ultimately determine the assembly and function of tissues and organs [11], [18], [19]. Moreover, ECM is indispensable for the maintenance of organ functions which are critically dependent on functional basal lamina layers, e.g. gas exchange in the lung and the glomerular filtration of the kidney.

Conventional in vitro kidney organ culture based on the filter-grid method after Trowell [20] has been applied to test various regulators of renal development, including ECM components and growth factors. Growth factors were added to the culture media and morphological changes of the organ were observed. The ECM components hyaluronic acid [21], collagen I [11], endostatin [22], matrix metalloproteinases [23] and growth factors including BMP-2, BMP-7 [24], pleiotrophin [25] and fibroblast growth factors [26], were demonstrated to influence branching morphogenesis and growth of explanted embryonic renal tissue. However, organ culture setups vary within studies. For example, different filter inserts and well plates are used, which results in varying amounts of media (400 μl to ml range). This makes it difficult to compare the studies since nominal concentrations of applied additives referred to solutions additionally added to these conventional settings (see Discussion). We wanted to analyze these previously tested ECM components in a single study and decipher the role of untested ECM components in kidney development. Our goal was to create a knowledge base for possible ECM-supported approaches in kidney tissue engineering and regeneration therapies. We used a recently established in vitro embryonic organ culture system [27] suitable for embryonic kidney rudiments (Fig. 1A and B) to study organ development. This system recapitulates many aspects of early in vivo development, like ureteric bud branching, nephron formation and clear anatomical cortico-medullary zonation combined with improved observability because of the transparent culture surface. The method uses a defined low media volume of 85 μl [27], as opposed to a ml range used in conventional embryonic kidney organ culture [20]. It allows manipulation of the cultured tissue, e.g. gene silencing, micro-dissecting and drug application as well as the chemical modification of the planar culture substrate, which is in our case glass in contrast to filter or metal grip of the conventional culture setups [20]. We have tested a broad range of ECM components and growth factors, previously considered to be involved in kidney development and repair (Table 1). These components have been reported to support nephron formation (BMP-7 [28], heparan sulfate [29], [30]) and the development of glomeruli (collagen IV [31], [32], laminin 8 [33], [34] and laminin 10 [35], [36]). Other factors that we tested have been found to interfere with branching morphogenesis and tubule formation (fibronectin [37], [38], heparin [29], [39], hyaluronic acid [HA] [21], the basal lamina element laminin 1 [40], nephronectin [41] and pleiotrophin which is particularly involved in the epithelial–mesenchymal communication [25]). In addition, we included components which were so far not found to be involved in or essential for kidney development, e.g. aggrecan, brevican, collagen I [42] which is an important factor for adhesion, morphology, growth, migration and differentiation in cell culture[43], as well as meteorin, neurocan, osteopontin [44], [45], tenascin-C [46] and vitronectin [47], [48]. The ECM components were presented to the developing organ either covalently immobilized on coverslips (Fig. 1C) or added to the culture medium at different concentrations (Fig. 1D), benefiting from the low volume used in our organ culture system, as we could apply high concentrations very cost effectively and ensure constant concentrations of additives during the whole culture period. Immobilized adult kidney ECM extracts obtained by decellularization [49] and reactive polymer films without any biomolecular components were included as reference controls.

Effects of the differently presented ECM components on the formation of organ phenotypes were systematically analyzed using a quantification of characteristic structural parameters of the developing kidney, such as number of ureteric bud tips, nephrons and overall size (Fig. 2). The results were summarized in an array-like overview, where we used a red–green color scheme to visualize relative differences to control kidneys.